Small interfering RNAs (siRNAs) are short, double-stranded RNAs (dsRNAs) that mediate efficient gene silencing in a sequence-specific manner. The specific cleavage of mRNA molecules targeted by siRNAs is mediated by the endogenous RNA interference (RNAi) pathway, which is present in most eukaryotic cell types. Using Drosophila melanogaster embryo lysates, Elbashir et al. identified siRNAs with 19-base-pair (bp) duplex regions and 2-nucleotide (nt) 3′ overhangs (the so-called 19+2 structure) as the most efficient triggers of sequence-specific mRNA degradation. The 19+2 siRNA structure has thus become the standard for designing gene-silencing RNA molecules for therapeutic applications.
Despite the significant promise of siRNA technology, several studies have demonstrated nonspecific effects triggered by conventional 19+2 siRNA structures. First, siRNA can silence non-target genes either by imperfect pairing between mRNA molecules and the antisense strand of siRNA or by incorporation of sense strand into RNA-induced silencing complex (RISC) that results in the cleavage of mRNAs complementary to the sense strand. Second, excess amounts of siRNAs can saturate the cellular RNAi machinery and competitively inhibit the activity of other siRNAs or microRNAs (miRNAs). Third, while siRNAs were originally designed to circumvent the dsRNA-induced innate immune response, several studies have reported that nonspecific innate immune response can be induced by siRNAs. These nonspecific effects triggered by siRNAs limit the development of siRNA as a therapeutic modality.
We have developed a novel siRNA structure termed asiRNA, which is an asymmetric shorter-duplex siRNA backbone structure with duplexes shorter than 19 bp. Importantly, this RNA duplex structure significantly reduces nonspecific effects caused by conventional 19+2 siRNA scaffold, such as sense strand–mediated off-target gene silencing and saturation of the cellular RNAi machinery.